Thermo Fisher Scientific KnockOut SR User guide
- Type
- User guide
KnockOut™ SR
Catalog Numbers 10828028, 10828010
Pub. No. MAN0007312 Rev. 6.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
Gibco™ KnockOut™ Serum Replacement (KnockOut™ SR) is a more defined, FBS-free formulation designed to directly replace fetal
bovine serum (FBS) in supporting the growth and derivation of human and other mammalian embryonic stem cells (ESCs) and induced
pluripotent stem cells (iPSCs) on fibroblast feeder cells. For human ESCs, KnockOut™ SR also supports cryopreservation, embryoid body
formation, and in vitro dierentiation studies. For mouse ESCs, KnockOut™ SR can replace FBS in media used for blastocyst injection,
embryo culture, electroporation or cationic lipid transfection, clone selection, cryopreservation, derivation of new ESC lines, embryoid
body formation, and in vitro dierentiation studies. By providing the documentation required for regulatory review, KnockOut™ SR enables
you to reduce your burden in qualifying reagents when transitioning from research applications to clinical applications. Each container is
sterile filtered.
Contents and storage
Contents Cat. No. Amount Storage Shelf life[1]
KnockOut™ Serum Replacement 10828010
10828028
100 mL
500 mL −20°C to −5°C. Protect from light. 18 months
[1] Shelf-Life duration is determined from Date of Manufacture.
Culture conditions
Cells: Mouse, human and other mammalian ESCs and iPSCs
Culture type: Adherent co–culture
Temperature range: 36°C to 38°C
Incubator atmosphere: Humidified atmosphere of 4–6% CO2
in air. Ensure proper gas exchange and minimize exposure of
cultures to light.
Procedural guidelines
• Refer to thermofisher.com for applications and protocols
using KnockOut™ SR, including detailed protocols for mouse
or human ESC/iPSC culture.
• KnockOut™ SR cannot be used as a replacement for FBS in
the plating of feeder cells.
• KnockOut™ SR does not contain trypsin inhibitors. Therefore,
trypsin must be removed or inactivated when culturing ESCs
or iPSCs in medium containing KnockOut™ SR.
• Due to the variability observed between pluripotent stem cell
(iPSC and ESC) lines, we recommend that you test dierent
lots of KnockOut™ SR to optimize your culture conditions.
Guidelines to prepare media
• Thaw KnockOut™ SR overnight at 4°C.
• Alternately, thaw KnockOut™ SR in a 37°C water bath with
frequent gentle swirling to expedite thawing.
IMPORTANT! Do not heat-inactivate.
• Occasionally flocculent material may be observed while
thawing. This material will go into solution with gentle
swirling at 37°C. Minimize dwell time in waterbath.
• KnockOut™ SR is stable for up to 4 weeks at 2°C to 8°C,
protected from light.
• Working volumes can be aliquoted and stored at −20°C to
−5°C. Thaw aliquots as needed. Avoid additional freeze-thaw
cycles.
• If using traditional DMEM for mouse ESCs/iPSCs, we
recommend using KnockOut™ DMEM instead.
Guidelines for use
• Complete medium is stable for 10 days when stored in the
dark at 2°C to 8°C.
• Avoid repeated warming and chilling of the complete
medium. Warm only the volume required for that day’s use.
• For best results, pre-equilibrate complete medium to
temperature (37˚C) and gases (5% CO2 in humidified air)
before use.
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.
• If lyophilized, reconstitute basic Fibroblast Growth Factor
(bFGF) and LIF Recombinant Mouse Protein, ESC-Qualified
to stock concentrations of 10 μg/mL in 0.1% bovine serum
albumin.
• GlutaMAX™ Supplement may be substituted with L‑glutamine
at the same molar concentration.
• Supplement complete medium with fresh 0.1 mM
2‑Mercaptoethanol immediately prior to equilibrating medium
to temperature and gases.
• If desired, antibiotics may be used.
Prepare complete media
Prepare complete media for human ESCs and iPSCs (Table 1)
or mouse ESCs and iPSCs (Table 2) cultured on mitotically
inactivated mouse embryonic fibroblasts (MEFs).
Table 1 Media for human ESCs/iPSCs
Reagent Stock conc. Final conc. For 100 mL
DMEM/F-12, GlutaMAX™
Supplement – 1X 79 mL
KnockOut™ SR – 20% 20 mL
NEAA[1] 10 mM 0.1 mM 1 mL
bFGF[2] 10 µg/mL 4 ng/mL 40 µL
2‑Mercaptoethanol[3] 55 mM 0.1 mM 182 µL
[1] Non-Essential Amino Acids
[2] basic Fibroblast Growth Factor
[3] Add immediately prior to use.
Table 2 Media for mouse ESCs/iPSCs
Reagent Stock conc. Final conc. For 100 mL
KnockOut™ DMEM – 1X 83 mL
KnockOut™ SR – 15% 15 mL
NEAA[1] 10 mM 0.1 mM 1 mL
GlutaMAX™ Supplement 200 mM 2 mM 1 mL
LIF Recombinant Mouse
Protein, ESC-Qualified 10 µg/mL 10 ng/mL 100 µL
2‑Mercaptoethanol[2] 55 mM 0.1 mM 182 µL
[1] Non-Essential Amino Acids
[2] Add immediately prior to use.
Note: KnockOut™ SR cannot be used as a replacement for FBS
in the plating of feeder cells. While the formulation contains
sucient factors to allow plating of ESCs and iPSCs, fibroblasts
have an increased need for undefined attachment factors and will
not adequately attach in this formulation. However, once plated,
the feeder cell layer will remain attached to the plates when
placed into media containing KnockOut™ SR.
Dissociate mouse ESC/iPSCs
For cell dissociation of mouse ESCs and iPSCs, we highly
recommend using StemPro™ Accutase™ Cell Dissociation
Reagent , as described in the detailed online protocol. Trypsin
may also be used. When dissociating ESC/iPSC clones with
trypsin following electroporation and selection, use soybean
trypsin inhibitor to quench trypsin activity.
1. Prepare a sterile 5 mg/mL solution of soybean trypsin
inhibitor in DPBS, no calcium, no magnesium.
2. Trypsinize clones and then add one-tenth the volume of
soybean trypsin inhibitor to the trypsinized ESCs/iPSCs.
3. Transfer cells to KnockOut™ SR supplemented medium and
replate.
ABC
Figure 1 Morphology of ESCs and iPSCs on MEFs
Morphology of C57BL/6 mouse ESCs A), H9 human ESCs B),
and human iPSCs C) cultured on MEFs in media containing
KnockOut™ SR.
2 KnockOut™ SR User Guide
Explanation of symbols and warnings
Temperature Limitation Manufacturer Batch Code Use By Catalog Number
Caution, consult
accompanying
documents
Consult instructions
for use Keep away from light Sterilized using aseptic
processing techniques Read Safety Data Sheet
Related products
Item Source
KnockOut™ DMEM (1X) 10829018
KnockOut™ DMEM/F-12 (1X) 12660012
DMEM/F-12, GlutaMAX™ Supplement (1X) 10565018
KnockOut™ SR−Multi-Species A3181501
A3181502
KnockOut™ SR Xeno-Free A1099201
KnockOut™ ESC/iPSC Media Kit A1412901
CF1 Mouse Embryonic Fibroblasts, Irradiated A34181
L-Glutamine, 200 mM (100X), liquid 25030081
25030024[1]
GlutaMAX™-1, 200 mM (100X) 35050061
35050038[1]
FGF-basic, Recombinant Human Protein 13256029
LIF Recombinant Mouse Protein, ESC-Qualified A35933
MEM Non-Essential Amino Acids Solution (100X) 11140076
11140068
2‑Mercaptoethanol (100X) 31350010[1]
StemPro™ Accutase™ Cell Dissociation Reagent A1110501
TrypLE™ Express Enzyme (1X), no phenol red 12604013
Trypsin-EDTA, (1X) 25300054
Trypsin Inhibitor, Soybean 17075029
DPBS, no calcium, no magnesium 14190144 [1]
[1] For European Customers Only
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and
Conditions of Sale found on Life Technologies' website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If
you have any questions, please contact Life Technologies at www.thermofisher.com/support.
KnockOut™ SR User Guide 3
Manufacturer: Life Technologies Corporation | 3175 Staley Road | Grand Island, NY 14072
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE,
MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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26 October 2020
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Thermo Fisher Scientific KnockOut SR User guide
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